All auditory information enters the brain at the cochlear nucleus, making this nucleus one of the first opportunities for modulation of incoming auditory information. Acetylcholine is an important neuromodulator present in the cochlear nucleus, and has been linked to processes such as gain adjustment, encoding of spectral peaks, selective auditory attention, and arousal. The role of cholinergic modulation in the cochlear nucleus is likely closely linked to the source providing the acetylcholine. Previously, it was believed that all acetylcholine in the ventral cochlear nucleus originated in the superior olivary complex; however a recent study found a cholinergic projection to the cochlear nucleus from areas in the midbrain associated with attention and arousal. Currently, little is known about this projection and how it might interact with other sources of cholinergic projections to the cochlear nucleus. This proposal focuses on the ventral division of the cochlear nucleus, and seeks to understand the circuitry of its cholinergic inputs. The first aim will identify the source of acetylcholine in the ventral cochlear nucleus. The second aim will associate cholinergic sources with cell types in the ventral cochlear nucleus. The third aim will identify connections between cholinergic inputs and specific output pathways of the ventral cochlear nucleus. These experiments will be accomplished using fluorescent and viral tracers to examine neurons projecting to and from the ventral cochlear nucleus. Immunochemistry will be used to determine whether tracer-labeled cells and terminals are cholinergic. Light microscopy will be used to find probable points of contact between cholinergic terminals and cells in the ventral cochlear nucleus, and electron microscopy will be used to confirm synaptic contacts. All experiments will be conducted in mice, as they are widely used in auditory research, and the methods proposed have been validated in this species. The results will provide important information about the sources of acetylcholine to the ventral cochlear nucleus and, by definitively linking cholinergic inputs to specific output pathways, will move one step toward understanding the roles of these inputs. These results have important implications for understanding normal auditory function, and for understanding pathological conditions. Acetylcholine in the cochlear nucleus has been demonstrated to play a role in hearing loss and tinnitus, making these results valuable to the study of both of these disorders. In addition to providing important scientific information, this proposal will allow training of the applicant in multiple anatomical techniques, experimental design, and light and electron microscopy as well as additional aspects of scientific career development such as grant writing and management.